POS1419 SINGLE CELL ANALYSIS REVEALING THE LECTIN BINDING PATTERN OF T-CELLS, NKT-CELLS, NK CELLS, B-CELLS AND MONOCYTE SUBSETS IN SYSTEMIC LUPUS ERYTHEMATOSUS
Authors: E. Szabo, G. Bodor, L. Kovács, G. Szebeni
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Cell surface glycosylation serves as a binding platform for endogenous lectin families such as galectins (Gals) or sialic acid immunoglobulin-like lectins (Siglecs) with well-known immunoregulatory functions. Several studies, including our own work <sup>[1]</sup>, have shown that Gals play an important role in pathogenesis of systemic lupus erythematosus (SLE) in animal models but their contribution to human SLE must be better elucidated. For these lectins to act, the appropriate glycan structures should be available on the target cells and should not be masked by alpha-2,6-sialylation which prevents binding of Gal-1, but not Gal-3. Although cancerous diseases are well-characterized by an altered glycome, limited information regarding cell surface glycosylation is gathered for autoimmune diseases like SLE. <h3>Objectives</h3> In the current study, we aim to analyse the ability of certain immune subsets to bind Gal-1, Gal-3, sialic acid binding C-type lectin (Siglec-1), the fucose binding <i>Aleuria aurantia</i> lectin (AAL), the sialic acid binding <i>Sambucus nigra</i> Agglutinin (SNA) lectin in SLE patients versus healthy controls. <h3>Methods</h3> We collected peripheral blood mononuclear cells (PBMCs) from 19 new or relapsing SLE patients with active disease (mean age: 47.5, SLEDAI-2K:15.8, anti-dsDNA level: 76.4 IU/ml) with no or minimal immunosuppressive therapy and 19 healthy age-matched controls. Multicolor antibody panel was designed to identify the main peripheral immune subsets: CD4+T-cells, CD8+ T-cells, CD4-/CD8- T-cells, CD4+/CD8+ T-cells; CD4+NKT-cells, CD8+NKT-cells, CD4-/CD8-NKT-cells, CD4+/CD8+NKT-cells; CD56+ NK cells; naive B-cells, memory B-cells, plasmablasts; classical monocytes, non-classical monocytes and intermediate monocytes. The five lectins (Gal-1, Gal-3, Siglec-1, AAL, SNA) were conjugated with different fluorophores for FACS. The binding of fluorochrome labelled lectins to each of these populations was assessed simultaneously by flow cytometry directly after thawing (resting state) and following activation. In case of T-cells, activation was achieved by a polyclonal activator (CytoStim), while B-cells and monocytes were activated by LPS. <h3>Results</h3> In our experiments with control PBMCs, titration and compensation of both antibodies and lectins were carried out and gating strategies were built to identify the above-mentioned immune subsets and their lectin binding characteristics. There were remarkable differences in lectin binding among different immune populations, e.g. classical and intermediate monocytes outperformed non-classical monocytes and lymphocytes in all lectin binding. Interestingly, naive B-cells were more sialylated (Median Fluorescence Intensity(MFI)<sub>SNA</sub>:2737) and bound less Gal-1 (MFI<sub>Gal-1</sub>:5730) than both memory B-cells (MFI<sub>SNA</sub>:2368; MFI<sub>Gal-1</sub>:16189) or plasmablasts (MFI<sub>SNA</sub>:1263; MFI<sub>Gal-1</sub>:16515). Similarly, highest SNA (MFI<sub>SNA</sub>:144) and lowest Gal-1 (MFI<sub>Gal-1</sub>:3658) binding was observed for CD4+ T-cells among the analysed T-cell subsets (MFI<sub>SNA</sub> range:12-120; MFI<sub>Gal-1</sub> range: 4201-5460) or NK cells (MFI<sub>SNA</sub> not detectable; MFI<sub>Gal-1</sub>:14288) or NKT-cells (MFI<sub>SNA</sub> range: not detectable-108; MFI<sub>Gal-1</sub> range:3580-6327). <h3>Conclusion</h3> The focus of this study is to identify relevant changes in the cell surface glycosylation of SLE immune subsets which is a unique approach and could contribute to a better understanding of the glycobiology of lupus. <h3>Reference</h3> [1]Deak M,Hornung A et al. <i>Immunobiology</i>. 2014;220(4):483-489 <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.